Identification of biomarker and development of screening method for kidney stone disease

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Date
2007
Authors
Lau, Wai Hoe
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Abstract
Kidney stone disease is the most common urological disorders that occurred in both men and women but with higher prevalence in men. The lifetime chance of an individual having a stone in kidney is approximately 10% and the risk of recurrence during 10 years period is 74%. Therefore, there is a great need to develop a screening method for detection of kidney stone disease. In this study, proteomic approach was used for extraction, separation and identification of urinary proteins from healthy subjects, stone formers and recurrent stone formers. Urinary proteins were extracted using salt precipitation technique and the protein extract was dissolved in solubilizing buffer. The mixture was separated according to their molecular weights using SDS-PAGE. The gel was then Coomassie Blue stained. The image of the gel was captured and analyzed by using an imaging system. The target protein bands were excised from the gel and proteins were digested In-gel by trypsin. The tryptic peptides were then eluted from the gel and analyzed using LC/MS/MS which allows amino acid sequencing of the analyzed peptides. The acquired MS/MS product ions spectrum was search against Mascot protein database search engine for protein identification. A total of twenty nine proteins were identified from healthy subjects, stone formers and recurrent stone formers. The urinary THP was identified as a biomarker for kidney stone disease and was confirmed by Western blotting. Employing THP as biomarker, SDS-PAGE and ELISA methods were developed for urinary THP quantification. For SDS-PAGE analysis, the urine sample was salt precipitated and concentrated ten times. The cut-off value of THP excretion by SDS-PAGE was 1.30 mg/mmol. However, urine sample for ELISA analysis was diluted ten times in sample buffer. The developed ELISA achieves linearity within the range of 109.33 ng/mL to 945.67 ng/mL of THP. In addition, the assay accuracy was around 98 – 101%. The assay precisions were less than 4% (C.V.) for repeatability and less than 5% (C.V.) for reproducibility. The assay specificity and sensitivity were 86% and 80%, respectively. Whilst the cut-off values of THP concentration by ELISA were 37.00 μg/mL and 41.20 μg/mL for male and female, respectively. SDS-PAGE shows a clearer cut-off between healthy subjects and stone formers as compared to ELISA. SDS-PAGE is a better method to quantify the amount of urinary THP.
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Master
Keywords
Science pharmacy , biomarker , screening method , kidney stone disease
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